Fig. 4

The effect of PKC on Nedd4–2 phosphorylation. (A, top panel): Phosphorylation of Nedd4–2. OAT1-expressing COS-7 cells were transfected with wild type Nedd4–2. Transfected cells were treated for 1 h, with 10 μM PMA in the absence or presence of 2 μM staurosporine (St). Cells were lysed and Nedd4–2 was immunoprecipitated (IP) with anti-FLAG antibody (epitope FLAG was tagged to Nedd4–2). Normal mouse IgG was used as non-specific binding control (IgG). Then immunoblotting (IB) was performed with anti-phospho-Ser/Thr primary antibody and anti-rabbit secondary antibody. (A, bottom panel): The same immunoblot from the top panel was stripped and reprobed with anti-FLAG antibody to determine the total amount of Nedd4–2 pulled-down. (B): Densitometry plot of results from (A) as well as from other repeat experiments. The values are mean ± S.D. (n = 3). *p < 0.05